3 Methods
3.1 Cell culture
3 Methods
3.1 Cell culture
3.1.1 General cell culture methods
MDBK cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10 % horse serum at 37°C and 5% CO2. The medium contains 60 μg/ml penicillin G and 50 μg/ml streptomycin. Confluent cells were passaged in a ratio of 1:20 every three to four days. Removal of the medium was followed by addition of 10 ml pre-warmed Versen trypsin, from which 9 ml were immediately removed. The cells were incubated with the remaining trypsin for 10-15 min at 37 °C to detach them from the surface of the 10 cm cell culture dish.
Afterwards 9 ml of medium was added and the cells were resuspended by up and down pipetting, before being moved to a fresh cell culture dish in the ratio described.
The medium was regularly tested by PCR and IF to exclude a contamination with BVDV and Mycoplasma. The horse serum was likewise proven free of BVDV and Mycoplasma and was inactivated prior to usage at 56 °C for 30 min.
3.1.2 Cryoconservation and recultivation of cells
For long-time storage at -150 °C, densely grown cells were trypsinized and collected in cell culture medium. After centrifugation (20 °C, 4 min, 805 x g) the supernatant was discarded and the cell pellet resuspended in fresh medium containing 10 % DMSO. The cell suspension was transferred into cryo tubes in a concentration of 2 x 106 cells/ml and set into a cell freezer at -80 °C for 24 h. Afterwards the cryo tubes were moved to -150 °C.
Frozen cells were recultivated by thawing at room temperature, followed by dilution in 14 ml fresh medium and centrifugation for 4 min (20°C, 805 x g) to remove the potential cytotoxic
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effect of DMSO. The supernatant was discarded and the cell pellet resuspended in fresh medium. The cell suspension was transferred to a 3.5 cm cell culture dish and medium was changed 4-6 h later. After the cells reached confluency they were trypsinized and transferred to a 10 cm cell culture dish.
3.1.3 Determination of cell number
After detaching and resuspending cells from the cell culture dish, a 1:10 dilution of the cells in 0.25 % trypan blue solution was prepared. Cells were counted in a Fuchs-Rosenthal counting chamber under the microscope. For an accurate determination of the cell number, four 1:10 dilutions of the cells were prepared and for each dilution eight squares were counted. The mean value of counted cells for one big square was inserted into the following formula:
∁= 𝑥̅ × 𝑓 ×1000 0.2 𝑚𝑙 C = cells per ml
f = dilution factor
𝑥̅ = mean value of counted cells for one big square
3.1.4 Virus infection
Infection of MDBK cells with BVDV was carried out with a specific multiplicity of infection (MOI). One requirement for an infection with a specific MOI is the accurate determination of the present cell number. For infections, cells were seeded 12-16 h before infection. For the determination of the present cell number at the infection time point, four additional wells or dishes were seeded. The cell number of these four wells or dishes was determined before the
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infection and the mean value of these counted cells was considered to be the number of cells at the time point of infection.
3.1.5 Virus propagation
For the propagation of virus, 2.3 x 106 cells were seeded 12-16 h before infection in a 10 cm cell culture dish. The infection was carried out with an MOI of 0.1. After inoculation for 1 h at 37 °C, 7.5 ml fresh medium was added to the virus suspension and the cells were incubated for additional 72 h. The supernatant was harvested, aliquoted and stored at -80 °C.
3.1.6 Endpoint titration and determination of viral titers
For the titration, 100 µl cell culture medium was pipetted into each well of a 96-well plate.
The virus was diluted 1:100 in cell culture medium and 50 µl of this dilution was added to the first lane of the 96-well plate. Afterwards 50 µl virus suspension was transferred from the first line of the 96-well plate to the second and respectively to the consecutive lines. Pipette tips were changed at each dilution step. Into each well 2.5 x 104 cells were pipetted and the plates were incubated for three days at 37 °C. Titration of virus stocks was performed three times in quadruplicates. Analysis of the titration was performed by immunofluorescence and the infectious viral titers were determined according to the following formula:
TCID50 (50 % tissue culture infectious dose) / ml = 3(𝑥+0.5)× 10 × 𝑓 x = mean value of highest virus-positive dilution step f = pre-dilution
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3.1.7 Indirect immunofluorescence
Since cp as well as ncp viruses were used in this study, analysis of infectious viral titers required indirect IF. The supernatant of the 96-well plates was removed and the plates washed one time with phosphate-buffered saline minus (PBSM). The cells were heat-fixated at 80°C for 4 h. Afterwards, cells were washed once with phosphate-buffered saline (PBS) and incubated with 50 µl of the monoclonal antibody (mAb) C16 at 37°C for 2 h in a dilution of 1:50. Following three consecutive washing steps, 50 µl of the secondary antibody Cy3-AffiniPure Goat Anti-Mouse IgG (H+L) was added at a dilution of 1:800 and incubated for 1 h at 37°C. The antibody was removed by three washing steps and distillated water was added to the wells. Both antibodies were prepared in the washing buffer containing PBSM + 0.05 % Tween 20.
The cells were analyzed under an inverted immunofluorescence microscope at a wavelength of 570 nm. Pestivirus infections was visible as red fluorescence located in the cytoplasm.
3.1.8 Viral growth kinetic
For the viral growth kinetic, 0.8 x 105 (39.5 °C) or 1.5 x 105 (33 °C) cells were seeded into one well of a 6-well plate 12 h before infection. The cells were incubated with CP7, NCP7, the ts mutants (TS42, TS43, TS45) and rts mutants (rTS42, rTS43, rTS45) at an MOI of 0.1 for 1 h at 33 °C or 39.5 °C. The inoculum was removed and cells washed six times with PBS.
To each well 2.5 ml fresh medium was added and the cells were incubated for 96 h at 33°C or 39.5 °C, respectively. At 0 h, 24 h, 48 h, 72 h and 96 h post infection (p.i.) 250 µl supernatant were collected and stored at -80 °C. After each sample collection, 250 µl fresh medium was substituted. The determination of infectious viral titers was performed by endpoint titration
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and indirect IF analysis as described above. The samples were undiluted (0 h p.i.) or diluted 1:10 (24 h p.i.) or 1:100 (48 h -96 h p.i.) prior to endpoint titration. Each sample was titrated one time in quadruplicates. In the viral growth kinetic analysis, the supernatants of three independent experiments were used.
3.1.9 Transfection of in vitro transcribed RNA
A day before the experiment, MDBK cells were splitted 1:3 to reach a confluency of 90 % at the day of electroporation. The media was discarded and the cells were washed with PBSM before they were trypsinized. Afterwards the cells were collected in PBSM and centrifuged (4 °C, 4 min, 805 x g). The supernatant was discarded and the cell pellet was resuspended in PBSM. The cells were counted and 5 x 106 cells were transferred into a 15 ml falcon. After centrifugation (4 °C, 4 min, 805 x g) the supernatant was discarded and the cell pellet resuspended in 260 µl PBSM. The transcript was added to the cell suspension and directly pipetted into a 2 mm electroporation cuvette. For electroporation an exponential protocol (180 V, 950 µF) was used. Afterwards the content of the cuvette was transferred in 10 ml fresh medium to a 10 cm cell culture dish and incubated at 37 °C. The medium was changed 4 h post transfection. Supernatant was harvested 72 h post transfection and stored at -80 °C.
3.1.10 Analysis of viral RNA synthesis
MDBK cells were infected with CP7, NCP7, the ts mutants (TS42, TS43, TS45) and rts mutants (rTS42, rTS43, rTS45) at an MOI of 1 and incubated at the stated temperature for 1 h. Afterwards the inoculum was removed and the cells were washed six times with PBS.
Fresh medium containing 10 % horse serum was added and the infected cells were incubated
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at the stated temperature. After removal of the supernatant and six washing steps with PBS, total cellular RNA was harvested 10 h p.i. and 24 h p.i. (only CP7 and NCP7). The RNeasy Mini Kit was used to isolate the RNA according to the manufacturer’s protocol (see chapter 3.4.3). The RNA was quantified via photometric analyses and 50 ng RNA was used in a qRT-PCR using the QuantiTect® SYBR® Green RT-PCR kit (see chapter 3.4.6). The primer pair OL_100 and OL_380 was used (see chapter 2.5). Changes in viral RNA synthesis are given in percentage based on the amount of viral RNA in cells infected with CP7 (= 100 %).
3.1.11 Drug treatment
The drug tunicamycin was used to induce the UPR. Cells were seeded 12 h before treatment.
The supernatant was removed and new medium containing tunicamycin in a concentration of 1 µg/ml or 2 µg/ml was added. Cells were incubated for 24 h at the specified temperature and lysated with the lysis buffer provided in the caspase assay kit of ABCam (see appendix) according to the manufacturer’s protocol.